Long-term specific tracing of the fibroblast activation protein (FAP) has been of great importance because it is heavily expressed by stromal fibroblasts of multiple diseases, and several disorders associated with FAP are chronical. Bioluminescence (BL) imaging has its advantages to detect FAP in vivo since no external excitation is required, but the current FAP-responsive BL probe was constructed by covalently masking the firefly luciferase substrate and easily secreted out from the animal, resulting in transient BL imaging of FAP. To circumvent this problem, a peptide-linked amphiphilic block copolymer-based probe (PABC) was developed and applied to the long-lasting BL image of FAP in vivo. For this purpose, an amphiphilic block copolymer containing an FAP-responsive peptide was fabricated to self-assemble into micelles, which act as a depot to load amounts of d-luciferin for constructing the BL probe. Upon reaction with FAP, the micelle would be destroyed to release the internal d-luciferin for BL emission by a luciferase-catalyzed reaction. By virtue of the high loading capability of micelles, the FAP was determined from 0.5 to 10 ng/mL with a detection limit of 0.105 ng/mL, and the high sensitivity makes the PABC capable of distinguishing cancer cells from normal ones. Importantly, compared with free d-luciferin, PABC can be used to persistently image the FAP in living cells and in vivo. This characteristic of long-lasting specific tracing of the FAP makes us envision that this BL probe could be used for screening of FAP inhibitors and diagnosing various FAP-related diseases in future.